Proteomic mapping in live
            <i>Drosophila</i>
            tissues using an engineered ascorbate peroxidase

Chen, Chiao-Lin; Hu, Yanhui; Udeshi, Namrata D.; Lau, Thomas Y. K.; Wirtz-Peitz, Frederik; He, Li; Ting, Alice Y.; Carr, Steven A.; Perrimon, Norbert

doi:10.1073/pnas.1515623112

Cited by 143 publications

(154 citation statements)

References 32 publications

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“…After trypsin digestion, peptides are labeled with iTRAQ or TMT reagents, differentially labeled samples are mixed and the mixed sample is desalted before injection on the MS. This protocol has been successfully used to map the mitochondrial matrix proteome in Drosophila muscle tissue 17 .…”

Section: Preparing Silac Samples For Ms Proteomics ● Timing Variable;mentioning

confidence: 99%

“…5). Since these two publications, the APEX proteomic method has also been used to map the mitochondrial matrix proteome in live Drosophila muscle tissue 17 , the proteome of endoplasmic reticulum (ER)-plasma membrane contact sites in HEK 293 cells 18 and the proteome of the primary cilium in inner medullary collecting duct epithelial cells 19 . We have also successfully used this methodology to map proteomes of cultured rat neurons (K.H.L., N.D.U., S.A.C.…”

Section: Introductionmentioning

confidence: 99%

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Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2

et al. 2016

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This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substrate, APEX2 generates biotin-phenoxyl radicals that covalently tag proximal endogenous proteins. Cells are then lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry. We describe the generation of an appropriate APEX2 fusion construct, proteomic sample preparation, and mass spectrometric data acquisition and analysis. A two-state stable isotope labeling by amino acids in cell culture (SILAC) protocol is used for proteomic mapping of membrane-enclosed cellular compartments from which APEX2-generated biotin-phenoxyl radicals cannot escape. For mapping of open cellular regions, we instead use a ‘ratiometric’ three-state SILAC protocol for high spatial specificity. Isotopic labeling of proteins takes 5–7 cell doublings. Generation of the biotinylated proteomic sample takes 1 d, acquiring the mass spectrometric data takes 2–5 d and analysis of the data to obtain the final proteomic list takes 1 week.

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Section: Preparing Silac Samples For Ms Proteomics ● Timing Variable;mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2

et al. 2016

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“…We also found abnormal, clustered mitochondrial phenotypes upon RNAi knockdown of marf and parkin in larval muscles (Wang et al, 2015) and we are currently using the larval musculature to identify new genes that genetically interact with parkin (Wang, et al, submitted). Of note, a recent paper highlights an additional feature of the Drosophila L3 musculature as a model in mapping the mitochondrial matrix proteome using an engineered ascorbate peroxidase (APEX) assay followed by mass spectrometry (Chen et al, 2015). …”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Analysis of mitochondrial structure and function in the Drosophila larval musculature

2016

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Mitochondria are dynamic organelles that change their architecture in normal physiological conditions. Mutations in genes that control mitochondrial fission or fusion, such as dynamin-related protein (Drp1), Mitofusins 1 (Mfn1) and 2 (Mfn2), and Optic atrophy 1 (Opa1), result in neuropathies or neurodegenerative diseases. It is increasingly clear that altered mitochondrial dynamics also underlie the pathology of other degenerative diseases, including Parkinson’s disease (PD). Thus, understanding mitochondrial distribution, shape, and dynamics in all cell types is a prerequisite for developing and defining treatment regimens that may differentially affect tissues. The majority of Drosophila genes implicated in mitochondrial dynamics have been studied in the adult indirect flight muscle (IFM). Here, we discuss the utility of Drosophila third instar larvae (L3) as an alternative model to analyze and quantify mitochondrial behaviors. Advantages include large muscle cell size, a stereotyped arrangement of mitochondria that is conserved in mammalian muscles, and the ability to analyze muscle-specific gene function in mutants that are lethal prior to adult stages. In particular, we highlight methods for sample preparation and analysis of mitochondrial morphological features.

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“…To discriminate between unlabeled peptides following a labeling event (Protein X and Y in Scheme 1) and nonspecific bead-binding proteins (Protein Z in Scheme 1), unlabeled peptides can be tagged with a different isotope composition [e.g., SILAC 4,5 or iTRAQ 6 ] during the sample preparation, and then peptides from a labeling event can be identified according to the expected isotopic ratio. However, this identification method based on unlabeled peptides has a caveat.…”

Section: Introductionmentioning

confidence: 99%

Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells

Lee

et al. 2016

ACS Cent. Sci.

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Mammalian target of rapamycin (mTOR) signaling is a core pathway in cellular metabolism, and control of the mTOR pathway by rapamycin shows potential for the treatment of metabolic diseases. In this study, we employed a new proximity biotin-labeling method using promiscuous biotin ligase (pBirA) to identify unknown elements in the rapamycin-induced interactome on the FK506-rapamycin binding (FRB) domain in living cells. FKBP25 showed the strongest biotin labeling by FRB–pBirA in the presence of rapamycin. Immunoprecipitation and immunofluorescence experiments confirmed that endogenous FKBP25 has a rapamycin-induced physical interaction with the FRB domain. Furthermore, the crystal structure of the ternary complex of FRB–rapamycin–FKBP25 was determined at 1.67-Å resolution. In this crystal structure we found that the conformational changes of FRB generate a hole where there is a methionine-rich space, and covalent metalloid coordination was observed at C2085 of FRB located at the bottom of the hole. Our results imply that FKBP25 might have a unique physiological role related to metallomics in mTOR signaling.

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Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

Cited by 143 publications

References 32 publications

Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2

Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2

Analysis of mitochondrial structure and function in the Drosophila larval musculature

Proximity-Directed Labeling Reveals a New Rapamycin-Induced Heterodimer of FKBP25 and FRB in Live Cells

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